1. Field of the Invention
Methods for detecting the presence of particular enzymes found in complex biological mixtures are required under a variety of circumstances. While it is usually possible to separate the proteins found in such a mixture into a plurality of fractions based on size, molecular weight, net electrical charge, and the like, it still remains necessary to detect the enzyme of interest in one or more of the separated fractions.
A number of methods for the detection of enzymes exist. For example, detection can be accomplished immunologically using a labelled antibody specific for the enzyme. Such immunological detection, however, is limited by the availability of suitable antibodies. Enzyme detection can also be accomplished by exposing a protein fraction (or other sample suspected of containing the enzyme) to substrate for the enzyme. The presence of the enzyme in the fraction or sample can then be determined based on conversion of the substrate to product. This latter approach, although useful in many circumstances, requires that the substrate and product be distinguishable in a convenient and rapid manner. Many times, however, convenient detection reactions are not available and the difficulty in distinguishing the substrate from product makes this latter approach for identifying enzyme activity impractical.
For the above reasons, it would be desirable to provide an enzyme detection method which provides for separation of product from the substrate prior to detection of the product. In particular, it would be desirable to provide such a method which allows for simultaneous screening of a plurality of samples.
2. Description of the Prior Art
A number of blot transfer techniques exist for analyzing proteins and polynucleotides. For example, Southern blotting provides for transfer of polynucleotide fragments on an electrophoresis gel to cellulose nitrate filter paper by blotting a buffer solution through the gel and the filter. The polynucleotide of interest is then located on the filter by detection with a complementary DNA or RNA probe. See, Southern (1979) J. Molec. Biol. 98:503-517. An analogous technique, referred to as the Eastern or Native blot, has recently been developed for protein analysis. The proteins are separated by electrophoresis in a native gel using a non-denaturing buffer. The proteins are then transferred from the gel onto nitrocellulose by blot transfer. The protein of interest may then be identified immunologically. The method has been described by Reinhart and Malamud (1982) Anal. Biochem. 123:229-235; and Smith and Summers (1980) Anal. Biochem. 109:123-129. Other blotting techniques include the Western blot for detecting protein under extremely denaturing conditions (see, Towbin (1979) Proc. Natl. Acad. Sci. USA 76:4350-4354), and Northern blots for the detection of RNA on specially prepared reactive paper (Alwine et al. (1979) Methods In Enzymology 68:220-242). None of these blot detection methods provides for detection of an enzyme product. Assays for enzymatic activity have been performed using ion exchange columns where at least one of the substrate or product is ionic. See 1984 Bio-Rad Catalog, pp. 19-20, and the references cited on page 20. The use of ion exchange chromatography in the detection of glutamate synthase is described in Somerville and Ogren (1980) Nature 286:257-259. Various techniques for separating and visualizing enzymes in native gels are described in Siciliano and Shaw (1976) in: "Chromatographic and Electrophoretic Techniques," Smith, ed., vol. 2, pp. 185-209, Wm. Heinemann Medical Books, Chicago Illinois, and Cardy et al., (1981) Techniques for Starch Gel Electrophoresis of Enzymes from Maize (Zea mays L.), Institute of Statistics Mimeograph Series No. 1317, North Carolina State University, Raleigh, North Carolina. The fluorescent staining technique employed in the Experimental section hereinafter for visualizing product is described in Davies and Miflin (1977) J. Chromatogr. 153:284-286.